DHT deficiency perturbs the integrity of the rat seminiferous epithelium by disrupting tight and adherens junctions.

In rats with a DHT deficiency induced by finasteride, morphological changes in the seminiferous epithelium were observed. The structural alterations were manifested by the premature germ cells sloughing into the lumen of seminiferous tubules. The etiology of this disorder could be connected with intercellular junctions disintegration. We showed in the immunohistochemical study the changes in expression of some proteins building tight and adherens junctions. The depression of N-cadherin, ß-catenin and occludin immunoexpressions could be the reason for the release of immature germ cells from the seminiferous epithelium. However, the observed increase of the immunohistochemical reaction intensity of vinculin, one of the cadherin/catenin complex regulators, could be insufficient to maintain the proper function of adherens junctions. The hormonal imbalance appears to influence the pattern of expression of junctional proteins in the seminiferous epithelium. It could lead to untimely germ cells sloughing, and ultimately could impair fertility.

Testis morphology in rats chronically treated with letrozole, an aromatase inhibitor.

The aim of this study was to investigate the influence of the long-term treatment of rats with letrozole on the testis morphology. The pharmacologically induced estrogen deficiency caused statistically significant decreases of both intratesticular and serum levels of estradiol, and morphological changes in the seminiferous epithelium and in the interstitial tissue of the testes. Six months of treatment resulted in the sloughing of premature germ cells of the seminiferous epithelium into the tubular lumen and in intraepithelial vacuolization. Multinucleated giant cells composed of premature germ cells, conglomerates of various cell nuclei and cell debris as well as irregularities and infoldings of the tubular basement membrane were also seen. Moreover, deep invaginations of the lamina propria with myoid cells were observed. Cells in the interstitial tissue showed changes similar to that observed in aging processes. The cytoplasm of LH-R-positive Leydig cells was loaded with lipofuscin granules. The number of lipofuscin-loaded cells was significantly increased in the interstitial tissue of testis in letrozole-treated rats. The results indicate the direct influence of estrogens on seminiferous tubules and the interstitial tissue morphology.

The expression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of rats with a dihydrotestosterone (DHT) deficiency.

In our previous studies, we showed that a finasteride-induced DHT deficiency may cause changes in the morphology of the seminiferous epithelium without any morphological alteration of the epididymis. In this study, we demonstrated the constitutive immunoexpression of inducible nitric oxide synthase (iNOS) in the testis and epididymis of Wistar rats treated with finasteride for 28 days (the duration of two cycles of the seminiferous epithelium) and 56 days (the duration of one spermatogenesis). We noted that a 56-day finasteride treatment mainly caused a decrease in the level of circulating DHT, as well as a statistically insignificant decrease in the level of T. The hormone deficiency also led to a change in the iNOS immnoexpression in the testis and epididymis of the finasteride-treated rats. In vitro, DHT did not modify NO production by the epithelial cells of the caput epididymis even when stimulated with LPS and IFNgamma, but it did give rise to an increase in NO production by the epithelial cells of the cauda epididymis without the stimulation. DHT did not have a statistically significant influence on estradiol production by cultured, LPS- and IFNgamma-stimulated epithelial cells from the caput and cauda epididymis. In conclusion, our data clearly indicates that a finasterideinduced DHT deficiency intensifies the constitutive expression of iNOS in most rat testicular and epididymal cells, so it can be expected that the expression of inducible nitric oxide synthase (iNOS) could be regulated by DHT. On the other hand, the profile of the circulating DHT and T levels strongly suggests that the regulation of constitutive iNOS expression is complex and needs more detailed study.

Rat epididymal epithelial cells and 17beta-estradiol synthesis under hCG stimulation in vitro.

Epithelial cells of human and animal epididymis display features of steroidogenic cells. Rat epididymal epithelial cells in vitro produce androgens which are converted to 17beta-estradiol, and released into the medium. The regulation of the epididymal steroidogenesis is not fully understood but it could be expected that it remains under LH influence. In previous study we observed that the morphology of rat epididymal epithelial cells in vitro was affected by hCG and the increase of amount of lipid droplets, glycogen and PAS-positive substances was observed. The present studies show the organelles which take part in synthesis of steroids in rat epididymal epithelial cells in vitro and the effect of hCG on E2 synthesis. The cells were cultured in the medium with/without DHT and without DHT in supplementation with hCG. After hCG stimulation the amount of an active mitochondria were increased when compared to the amount of mitochondria in the epididymal epithelial cells cultured without DHT. Ultrastructure of the cells was similar to the cells cultured with DHT, while the cytoplasm of the cells cultured without DHT was disorganized. The synthesis of 17beta-estradiol was stimulated by hCG, that exerted its effect through LH/hCG receptors, localized in the epididymal epithelial cells.

Hormonal status of male reproductive system: androgens and estrogens in the testis and epididymis. In vivo and in vitro approaches.

The purpose of this article was to summarize our results on the role of androgens and estrogens in human, rodent and equine testes and epididymides, in both, physiological and patological conditions, obtained in the space of the Solicited Project (084/PO6/2002) financially supported by the State Committee for Scientific Research during the last three years. Testosterone produced by Leydig cells of the testes is clearly the major androgen in the circulation of men and adult males of most mammalian species. However, androgen metabolites make up a significant fraction of total circulating steroids. Moreover, androgen metabolism may proceed to amplify the action of testosterone through its conversion to dihydrotestosterone (DHT) or its aromatization to estradiol. The distribution of androgen and estrogen receptors (ARs and ERs) within male reproductive tissues is important because of their crucial role in mediating androgen and/or estrogen action. Attempts were undertaken to discuss not only the role of aromatase and ERs in mediating the action of estrogens in the male, but also the importance of DHT in hormonal regulation of the epididymis. In the latter, alterations caused by finasteride treatment and lead-induced oxidative stress are described. Male reproductive function of the testis and epididymis reflected by the alterations in enzymatic activity, distribution of steroid hormone receptors, differences in steroid hormone levels and altered gene expression of antioxidant enzymes are also discussed.

Immunolocalization of androgen receptor in the epididymis of rats with dihydrotestosterone deficiency.

Dihydrotestosterone (DHT), 5alpha-reduced metabolite of testosterone, is the most potent androgen in the epididymis. The conversion of T into DHT is carried out by 5alpha-reductase. The activity of 5alpha-reductase type 2, preferentially expressed in the epididymis can be inhibited by a finasteride (a steroid-based specific inhibitor of 5alpha-reductase type 2) which results in DHT deficiency. The aim of the study was to examine the morphology of epididymis and the immunolocalization of an androgen receptor (AR) in the initial segment, caput and cauda epididymis of rats treated with finasteride for 56 days. There were no morphological changes in the morphology of epididymal epithelium in the experimental rats. Immunostainable AR was localized in nuclei of epithelial cells, smooth muscle cells and mainly in the cytoplasm of interstitial cells in the epididymis of control rats. In the epididymis of experimental rats, AR immunostaining was noticed mainly in the cytoplasm of epithelial cells and interstitial cells. The single cells of the initial segment epithelium, basal cells and smooth muscle cells of cauda epididymis showed nuclear AR staining. In conclusion, finasteride affected the expression of the AR in the rat epididymis without changing the morphology of epididymal epithelium. Altered AR expression reflected the hormonal status within the epididymis.

Effect of quercetin on experimental hyperlipidemia and atherosclerosis in rabbits.

Cardiovascular disease is currently the leading cause of death in the West, and a search for factors limiting its occurrence is ongoing. Accumulated data indicate that quercetin, the major flavonol in the plant kingdom, may possess beneficial effects in atherosclerosis. The present study aimed at determination of effects of quercetin on hyperlipidemia and development of atherosclerotic lesions in two animal models, i.e. diet induced hyperlipidemia and aortic atherosclerosis, and in injured carotid artery in rabbits fed high-fat diet for 12 and 4 weeks, respectively. It was demonstrated that quercetin was effective in reducing serum triglycerides and cholesterol levels elevated by high-fat diet, after 12 weeks of the experiment. This activity was less prominent in the 4-week study in injured carotid artery rabbit model. Hypolipemic properties of the flavonoid were associated with the reduced formation of atherosclerotic plaques, both in the aorta (12-week study) as well as within injured carotid artery (4-week study) in high-fat diet-fed animals. The surface of the intima covered with atherosclerotic plaques in high-fat diet-fed rabbits was 24.6 +/- 33.1% in comparison to 0.7 +/- 1.3% (p < 0.05) in quercetin and high-fat diet supplemented animals. It is evident from the present study that quercetin possesses both hypolipemic and antiatherogenic properties.

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis.

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.

Localisation of oestrogen receptors (ERalpha and ERbeta) in the human and rat epididymides.

The aim of the study was to compare the localisation of oestrogen receptors alpha and beta (ER alpha and ER beta) in the human and rat epididymides. In the human epididymis the immunoexpression for ER alpha was detected in the nuclei of the caput epithelial cells, while positive reaction for ER alpha was observed in the nuclei of the cauda epithelial cells. In the rat epididymis, immunoexpression for ER alpha showed nuclei of the caput and cauda epithelial cells. However, the reaction was stronger in cells of the caput epididymis. A positive reaction for ER beta was observed in the nuclei of smooth muscle cells of the epididymal duct and in the nuclei of interstitial tissue cells of the rat caput and cauda epididymis. We have demonstrated that localisation of ER alpha and ER beta is cell-, region- and species-dependent.

Effect of hCG on the morphology of rat epididymal epithelial cells in vitro.

The epididymis is an androgen-dependent organ. The hormones regulate the morphology and secretory activity of the epididymal epithelial cells. The cells in vitro resume their function as in vivo and also reveal features of steroidogenic cells. It can be expected that, as with Leydig cells, the morphology and function of the cells can be regulated by LH/hCG. The aim of the study was to assess the morphology of epididymal epithelial cells in vitro after stimulation with hCG. The experiment was performed on cells isolated from sexually mature rats. The epididymal epithelial cells were cultured in a medium with the addition of dihydrotestosterone (DHT). Moreover, the cells were cultured in the medium with DHT and without DHT but enriched with hCG. The epididymal epithelial cells cultured with DHT formed a monolayer and accumulated glycogen, a PAS-positive substance and lipid droplets. The cells cultured without DHT were stellate in shape and low in glycogen and PASpositive substance but they contained lipid droplets. The morphology of epididymal epithelial cells cultured without DHT but after stimulation with hCG was similar to the morphology of the cells cultured with DHT. This was the first sign that the morphology of the cells can be influenced by hCG.